Project Summary/Abstract The Type 1-interferon (IFN-?/?) response is the first line of defense against viral infections. Hundreds of proteins, referred to as ISGs (Interferon-Stimulate Gene products) are synthesized in response to IFN-? or ? stimulation, and these proteins mediate an extremely broad spectrum of anti- viral effects. ISG15 was one of the first ISGs to be identified and the first ubiquitin-like modifier (Ubl) to be discovered, however it is only recently that we begin to have an understanding of the remarkable biochemical functions of this protein. ISG15 has anti-viral activity against a wide range of human viruses, including Influenza, Herpesviruses, Retroviruses, Sindbis, and Ebola. ISG15 is conjugated to hundreds of cellular proteins in IFN-stimulated cells, and a single IFN-induced ligase, Herc5, mediates conjugation to these target proteins. Herc5 is ribosome-associated and ?ISGylates? newly synthesized protiens in a co-translational manner, with little or no target protein specificity. We hypothesize that in the context of an interferon response, newly translated viral proteins, rather than cellular proteins, are the primary targets of this system, and that ISG15 acts by sterically interfering with the function of viral proteins, particularly those that form oligomeric structures (such as capsid proteins). Surprisingly, ISG15 has a second function as an extracellular protein that stimulates the production of Type 2 Interferon, IFN-?. While this function was reported over two decades ago, there was little follow-up to the original studies. This function returned to the fore with the recent discovery of ISG15-deficient patients who were highly susceptible to mycobacterial infection as a result of being unable to mount an effect IFN-? response. Further characterization of this activity has shown that ISG15 is highly synergistic with IL-12 in stimulating production of IFN-? by Natural Killer (NK) cells. This project will address critical biochemical questions in both of the ISG15 functions described above ? the intracellular conjugation of ISG15, and the extracellular IFN-? signaling by ISG15. In the first aim we will characterize the factors that localize Herc5 to the ribosome, identify the determinants that confer ISG15 ligase activity to Herc5, and gain insights into Herc5 activities through characterization of an unusual viral inhibitor of ISGylation. In the second aim we will characterize the determinants within the ISG15 protein required for IFN-? signaling, identify the putative cell surface receptor for ISG15 receptor, and characterize the down-stream ISG15 signaling events. We anticipate that these biochemical studies will provide important insights into the innate immune response to both human viral and microbial infections.